Open AccessDirect adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase
Author(s) -
Zhining Yang,
Chengliang Zhang,
Lian Guojun,
Shen Dong,
Menghui Song,
Hengrong Shao,
Jingmei Wang,
Zhong Tao,
Zhenni Luo,
Shengnan Jin,
Chunming Ding
Publication year - 2022
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gkac604
Subject(s) - rna ligase , biology , dna ligase , oligonucleotide , adenylylation , enzyme , rna , biochemistry , polynucleotide , polynucleotide phosphorylase , nucleotidyltransferase , microbiology and biotechnology , dna , biosynthesis , gene , purine nucleoside phosphorylase , purine
5'-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5'-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.